Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington’s disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates. Display omitted •Genome-wide RNAi screen identified 20 lincRNAs controlling pluripotency•lincRNA TUNA is required for pluripotency and neural differentiation•TUNA interacts with RNA-binding proteins through a conserved sequence•TUNA expression in Huntington’s patients was associated with disease grade Using a genome-wide shRNA library targeting lincRNAs, Lin et al. performed an unbiased loss-of-function study in ESCs and identified 20 lincRNAs involved in pluripotency maintenance. A lincRNA, TUNA, forms a multiprotein complex with the RNA-binding proteins PTBP1, hnRNP-K, and Nucleolin. TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function in adult vertebrates.